PROPOSAL SUMMARY Discovery of endogenous stem cells found within the heart, cardiac progenitor cells (CPC), has prompted intense basic discovery in multiple experimental animal models and clinical trials in heart failure patients. A survey of the literature reveals that the most popular experimental animal models exhibiting regenerative properties are also characterized by genome duplication or polyploidy. Our lab has recently discovered a fundamental difference between human and rodent CPCs: rodent CPCs possess polyploid mononuclear tetraploid (4n) chromatin content, whereas human CPCs are mononuclear diploid (2n) cells. This fundamental biological distinction between humans and rodents prompts provocative questions regarding regenerative potential differences between humans versus other species as well as the translational applicability of regenerative studies performed in rodent models. If ploidy is an integral aspect of tissue regeneration in lower vertebrates and other species, then elucidating the biological basis of CPC ploidy and mechanistic differences in cell signaling and mitosis between polyploid rodent CPCs versus diploid human CPCs will provide important insight for enhancement of regenerative potential. The overarching hypothesis of this proposal is that mononuclear chromatin duplication in CPCs improves regenerative capacity of the heart by overriding senescence-induced cell cycle arrest and increasing tolerance of DNA damage and oxidative stress. The short-term goal is to establish biological distinctions and elucidate unique molecular properties of polyploidy CPCs relative to diploid human CPCs. The significance is to understand the advantages of polyploidy for regeneration while also uncovering previously unrecognized limitations of extrapolating from experimental animal model studies to clinical interventional approaches. Two specific aims are proposed based upon the following hypotheses: (1) Chromatin content in murine CPCs responds to alterations in environment leading to changes in gene transcription and mitotic chromosomal alignment, and (2) CPCs of murine origin with tetraploid content possess enhanced regenerative characteristics and potential relative to diploid CPCs by phenotypic analysis as well as following adoptive transfer into an experimental infarction injury model. The novelty and impact is to define novel biological attributes of a well-known and heavily studied cardiac stem cell and to apply that understanding to elucidate the molecular and cellular basis of regenerative responses in human versus murine myocardial responses to pathologic injury. The long-term goal is to apply the knowledge gained from understanding the role of polyploidy in regeneration to improve upon clinically relevant therapies in the treatment of heart failure.